1. Field of the Invention
The field of the invention is improved dry reagents for instrumented whole blood tests useful for diabetics.
2. Description of the Related Art
This application claims the priority benefit of provisional patent application No. 60/327,535 “Dual glucose-hydroxybutyrate analytical sensors”, filed Oct. 5, 2001.
Blood glucose monitoring has revolutionized the treatment of diabetes. Large-scale clinical trials have clearly demonstrated that frequent blood glucose monitoring can aid in the prevention of many of the long-term complications of diabetes, such as diabetic retinopathy, circulatory disorders, and death. After nearly twenty years of development, blood glucose monitoring has now become a several billion dollar a year business.
As the blood glucose-monitoring field has advanced, the various blood glucose monitors have become more and more generic. All possess good accuracy, ease of use, and speed. As a result, the various manufacturers of blood glucose monitors have focused major efforts on gaining minor technical advantages to make minor improvements in their respective market shares. Such improvements may include minor improvements in speed, blood sample size, ease of sample application, cost, etc. All, however, produce test strips that measure only blood glucose.
Although blood glucose is clearly the most important biochemical parameter to measure in diabetes, it is not the only parameter of medical interest. Other parameters of medical relevance include glycosylated hemoglobin, used to measure long-term blood glucose control, and ketone levels, used to indicate if the patient is at risk for diabetic ketoacidosis.
Diabetic ketoacidosis is a major complication of diabetes. Such conditions occur during times of extreme insulin deficiency. Here the diabetic's tissues are unable to process glucose, and as a result, initiate the biochemical processes that result in the formation of ketones and excess blood glucose. During periods of insulin starvation, body cells are unable to metabolize glucose as an energy source and instead metabolize fat as an energy source. Ketone bodies, made up of acectoacetate, acetone, and beta-hydroxybutyrate (also called D-3-hydoxybutyrate) are produced from this fat metabolism process, and these build up in the blood. Excessive levels of ketone bodies in turn can alter the pH balance of the blood to a more acidic state, as well as other undesirable complications, eventually leading to confusion, coma, and death. In the early stages of fat metabolism, the ketone bodies contain relatively large amounts of acectoacetate and acetone. However in more profound ketoacidosis, the ketone bodies contain primarily beta-hydroxybutyrate.
Each year, about 12 out of every 1000 diabetics are hospitalized for Ketoacidosis, and 2% of those hospitalized die from it. It is the commonest cause of death for diabetic children.
Early detection is the best way to prevent diabetic ketoacidosis. If detected in time, rehydration and low-dose insulin therapy can be used to treat ketoacidosis. Thus means to ensure that the onset of ketoacidosis is promptly detected are of extreme utility to diabetics.
Means to measure ketone levels are known in the art. These include visually read test strips for acetone or acectoacetate in the urine, as well as whole blood tests for beta-hydroxybutyrate. Diabetics are trained that whenever their glucose levels are high, they should follow up by immediately running a separate ketone test.
Examples of urine ketone dry reagent tests include Ketostix, Keto-Diastix (Beyer) or Chemstrip K (Roche). Such urinary tests generally use non-enzymatic detection methods (such as nitroprusside based chemistries) that are primarily sensitive to acectoacetate, slightly sensitive to acetone, and not at all sensitive to beta-hydroxybutyrate. One drawback of tests that measure only urinary acectoacetate or acetone is that such tests can miss or underreport extreme levels of ketoacidosis. In mild ketosis, the body produces acectoacetate, acetone and beta-hydroxybutyrate in relatively proportionate amounts, and thus urinary tests for acectoacetate and acetone will detect mild ketosis. However in extreme ketoacidosis, the body produces mostly beta-hydroxybutyrate and relatively small amounts of acectoacetate and acetone. Thus non-enzymatic nitroprusside based acectoacetate and acetone sensitive tests may become insensitive to extreme ketoacidosis right when they are needed the most.
Simple dry reagent whole blood tests for beta-hydroxybutyrate, the most clinically relevant indicator of ketoacidosis, are known in the art. Presently, such dry reagent tests use a disposable reagent that performs only the beta-hydroxybutyrate test. Often this disposable beta-hydroxybutyrate reagent is read in a meter that is capable of reading a number of different types of single test reagents. For example, GDS diagnostics, Elkhart Ind., sells the “Stat-Site™” meter, which can read separate colorimetric dry reagent tests for either whole blood glucose or ketones (beta-hydroxybutyrate). This technology is taught in U.S. Pat. No. 5,139,685. Polymer Technology Systems of Indianapolis Ind. sells the Bioscanner™ meter that can also read separate calorimetric dry reagent tests for either whole blood glucose or ketones. Similarly, MediSense sells the “Precision Xtra™” meter that can read separate electrochemical dry reagent tests for either glucose or beta-hydroxybutyrate.
Other one-meter-multiple-reagents systems are in commercial use. The LXN Corporation sells the Duet™ and “In Charge System™” meters that are capable of reading either a calorimetric glucose dry reagent test, or alternatively a calorimetric glycated protein (fructosamine) dry reagent test. These are discussed in more detail in U.S. Pat. Nos. 5,695,949 and 6,027,692.
Although diabetics are accustomed to testing their blood glucose several times a day, they may often forget to run a ketone test, since such tests require extra reagents and effort. Indeed, in an effort to correct for this normal human lapse, some glucose meters, such as the LifeScan “ultra” blood glucose system, will attempt to remind users to run ketone tests by an extra “Ketones?” meter prompt. However, clearly many diabetics will ignore this reminder.
Ideally, what is best from a medical perspective is a blood glucose test that automatically (without any extra user thought, process, or intervention) also reports blood beta-hydroxybutyrate levels using the same drop of blood used to perform the standard and habitual glucose test. Indeed such a combined test would save many lives by facilitating the early detection of ketoacidosis. Additionally, such combined tests would be of strong commercial interest as well, since if everything else were equal, a combined glucose/beta-hydroxybutyrate test would be strongly preferred by diabetics over the glucose-only tests presently used.
However no such single blood drop activated, combined blood-glucose/blood-beta-hydroxybutyrate dry reagent has previously been proposed, invented, or commercialized.
By contrast, combined glucose—ketone test strips have been available for urine testing for many years. Given the competitive nature of the blood glucose-monitoring field, why does this discrepancy exist between the long-term commercialization of combined urine glucose-ketone dry reagent test strips, and the complete lack of any prior art in combined whole blood glucose/beta-hydroxybutyrate dry reagent tests?
The difference is almost certainly due to the radically different nature of the two different sample types. Urine is available in large (100+ milliliter [ml]) quantities. It is nearly transparent. Thus a combined glucose—ketone dry regent test may be made by simply putting a calorimetric glucose dry reagent test pad onto solid support a certain distance away from a colorimetric ketone dry regent test pad. Because large amounts of sample are present, the distance between the two test pads can be so great as to minimize any “cross talk” due to reaction intermediate or colorimetric dye indicator diffusion between the two pads.
It is often the case in nearly every area of technology that devices optimized for a single purpose outperform devices optimized for multiple purposes. Blood glucose testing has been a mature field for nearly twenty years, and blood glucose meters and reagents have evolved to a highly advanced state. Patients and physicians are unlikely to accept a dual glucose—beta-hydroxybutyrate reagent as being a genuine improvement unless, at a minimum, the glucose portion of the reagent performs at a level that is competitive with stand-alone blood glucose tests. If the combined reagent requires no extra user effort, the blood glucose portion is competitive, and the extra cost for the secondary function is minor, then the user will benefit and the combined reagent will likely be a medical and commercial success.
In this context, the commercial success of combined urine—ketone test strips can be understood. These devices function with the same urine sample and require no additional user effort. The urine blood glucose part of a combined urinary glucose-ketone test strip performs as well as stand-alone urine blood glucose test strip.
By contrast, combined whole blood glucose—beta-hydroxybutyrate dry reagents must overcome some formidable technical challenges. Whereas urine samples typically have a volume of 100 ml (milliliters), blood samples, typically derived from a fingerstick, are more typically have a volume around 1–10 ul, (microliters). This is nearly five orders of magnitude less in size. Whereas urine is nearly transparent and relatively free of optical and electrochemical interfering substances, blood is intensely colored and contains nearly 50% hemoglobin and other strong optical and electrochemical interfering substances.
In order to meet the requirement for no additional user effort, a whole blood combined glucose—ketone/beta-hydroxybutyrate test must place both the glucose sensing means and the ketone/beta-hydroxybutyrate sensing means close enough together as to both be activated with the same small (1–10 ul) drop of whole blood. Further, the test must be designed to minimize “cross talk” between such closely spaced sensing means.
3. Prior Art
Visually read beta-hydroxybutyrate sensors and ketone sensors.
U.S. Pat. No. 4,147,514 teaches a urine test strip for detecting urinary acetone and acetoacetic acid by means of an improved nitroprusside reaction. This urinary ketone test strip patent, in conjunction with U.S. Pat. No. 3,814,668 for a urinary glucose test strip, forms the basis for the popular Keto-Diastix® Reagent strips for urinalysis, produced by Bayer Corporation, Elkhart Ind.
U.S. Pat. No. 4,397,956 teaches a whole-blood modification of the combined urine glucose—non-enzymatic ketone test strip. In this modification, a separate glucose reagent pad and separate ketone pad are mounted on the same support. Both pads are covered with a blood separation coating. Two drops of blood, one for each separate reagent pad, are applied to the device. The user manually times the reaction by allowing the blood to soak in for one minute, and then manually wipes or washes off the excess blood from the outer layer of the pad.
As taught, the device of U.S. Pat. No. 4,397,956 measures whole blood acetoacetate using the sodium nitroprusside reaction, rather than the preferred enzymatic beta-hydroxybutyrate specific reaction. Thus the test reagent of U.S. Pat. No. 4,397,956 would be expected to suffer from the previously mentioned beta-hydroxybutyrate insensitivity clinical deficiencies of this type of reaction chemistry. This clinical deficiency, on top of other test deficiencies such as the requirement for multiple blood sample application steps, and extensive user intervention (timing, washing) teaches against the need for a competitive and automated dual glucose/beta-hydroxybutyrate whole blood test.
Prior art for single analyte glucose electrochemical sensors can be found a variety of patents, including many assigned to Genetics International, Medisense, E. Heller, & Company, Therasense, Selfcare, Boehringer Mannheim, and others. These include U.S. Pat. Nos. 4,545,382; 4,711,245; 4,758,323; 5,262,035; 5,262,305; 5,264,105; 5,286,362; 5,312,590; 5,320,725; 5,509,410; 5,628,890; 5,682,884; 5,708,247; 5,727,548; 5,820,551; 5,951,836; 6,134,461 and 6,143,164;
Prior art for single analyte hydroxybutyrate electrochemical sensors was published by Batchelor, et. al, “Ampherometric assay for the ketone body 3-hydroxybutyrate” Analytica Chimica Acta 221 (1989), 289–294.
U.S. Pat. No. 4,225,410 discloses an integrated array of electrochemical sensors where each sensor is a complete self-contained electrically isolated electrochemical cell, mounted on a solid support that contains a plurality of such cells. As is the case for previous art covering multiple colorimetric reagent pads on a single solid phase support, placing multiple electrically isolated electrochemical cells on a single solid phase support is also unsuitable for small rapid, low cost, analysis of 1–10 ul volume whole blood samples. Due to the surface tension characteristics of blood, separation of a single 1–10 ul droplet of whole blood into multiple electrically isolated droplets must overcome surface tension effects, and thus is energetically unfeasible without the intervention of energy added by some extra mechanisms. Although such mechanisms are known in the art (e.g. U.S. Pat. No. 6,090,251, etc.), the extreme manufacturing cost sensitivity of practical blood glucose tests should be recognized. Any commercially practical dual-purpose glucose—beta-hydroxybutyrate electrochemical sensor must be price competitive with mass marketed single purpose glucose sensors, which can typically be produced at costs of about 10–20 cents per sensor. This brutal economic constraint on manufacturing costs eliminates all but the simplest combined designs from consideration. At the present state-of-the art, it appears unlikely that means will be found to mass produce, for a total cost of 10 to 20 cents per unit, a fully functional combined purpose electrode-containing-reagent, that also contains extra mechanisms to reliably and almost instantly separate a microlitre sized drop of blood into two or more electrically isolated droplets.
Prior art for electrically triggered optical test reagents includes U.S. Pat. Nos. 5,344,754 and 5,554,531.
Prior art for fiber optical biochemical sensors includes U.S. Pat. No. 4,682,895, which teaches fiber optical probes with sharp, 180 degree bends at the sensor tip. Other prior art includes U.S. Pat. No. 4,548,907, which teaches bifurcated optical probes for use with pH dependent fluorophores.